Compound and device for treating bone and/or cartilage defects

ABSTRACT

The present invention relates to compositions devices and methods for treating bone and/or cartilage defects, and a method for manufacturing such a composition or device. In a certain embodiment, the invention provides a device and/or composition for treating bone and/or cartilage defects, having at least one collagen, for example of animal origin, and further containing at least one substance having an osteo-inductive or chondro-inductive activity, at least one differentiation and/or growth factor having osteo-stimulative and/or chondro-stimulative effect, and at least one filling material, in which the composition is in the form of a lyophil.

RELATED APPLICATIONS

The present application is related to German patent application number10 2007 012 276.6 filed in the German Patent Office on Mar. 8, 2007,U.S. provisional application Ser. No. 60/993,146, filed Sep. 10, 2007 inthe U.S. Patent and Trademark Office, and PCT applicationPCT/EP2008/001604, the contents of each of which is hereby incorporatedherein in its entirety.

TECHNICAL FIELD

The present invention provides compositions, devices, and method ofmanufacturing thereof; and methods for treating bone and/or cartilagedefects.

BACKGROUND

Natural bone is made up of a combination of inorganic elements, organicelements, and water. Inorganic elements, for example hydroxyapatite,make up approximately 70% of natural bone. Organic elements such ascollagen type I make up approximately 20% of natural bone, and watermakes up approximately 10%. Bone-forming cells, for example osteoblasts,produce and secrete or bind the organic elements. Further, non-collagenproteins, in particular osteocalcin, osteonectin, osteopontin, cytokine,growth inducing factor, proteoglycan and fat are present in naturalbone.

Natural bone is constantly being degraded and regenerated, i.e.re-modelled. Osteogenesis, i.e., bone regeneration, originates from thebone itself, as observed for example during healing of bone fractures.Thus, natural bone has the capability to generate new functional bone.

In many cases, however, regeneration of natural bone is restricted. Thismay be caused by, for example, the extent of a bone defect or bydifferent functional disorders, for example metabolic disturbances.Treatment of bone defects remains an important field, for example inorthopedics, repair to jaws, teeth and hands, surgery in connection withaccidents, and facial surgery.

Bone defects have different origins, caused for example by cysts,atrophies, or tumors. Complicated comminute fractures, congenitaldeformities and loosening of implants also cause bone defects. There isincreasing focus on the reconstruction of bone defects using suitablematerials.

Bone graft substitute products on the market include InFuse®manufactured by Medtronic Sofamor Danek (Minneapolis, Minn.), and OP-1®Putty manufactured by Stryker Corporation (Kalamazoo, Mich.). Theseproducts are sold as kits that separately contain a bovine collagenscaffold and human recombinant Bone Morphogenic Protein (“rhBMP”) in avial.

InFuse® uses rhBMP-2 which prior to implantation is soaked onto asponge. OP-1® Putty is provided as two components: a vial containing onegram dry powder of bovine collagen, OP-1®, rhBMP-7, aid another vialcontaining a putty additive of carboxymethylcellulose dry powder. Bothcomponents must be combined with saline to produce the implant.According to the OP-1® Putty package insert, 96% of patients treatedwith OP-1® Putty develop an immune response to the product.

With each of these products, the rhBMP component is applied to thescaffold is performed during the surgical procedure. Applying the rhBMPsolution to the surface of the scaffold potentially results in aheterogeneous distribution of the rhBMP, i.e., certain areas of thescaffold will have a higher concentration of rhBMP than other areas ofthe scaffold. Further, as the surgeon is required to assemble theproduct in the operating room, there exists a risk of infection andinconsistent dosage from procedure to procedure.

There is a need for sterile homogeneous devices and compositions withhigh osteo-inductive or chondro-inductive activities and methods fortreating bone and/or cartilage defects.

SUMMARY

The invention in one embodiment provides a composition for treating boneand/or cartilage defects, having at least one collagen, preferably ofanimal origin, and preferably containing at least one substance havingan osteo-inductive or chondro-inductive activity, at least onedifferentiation and/or growth factor having osteo-stimulative and/orchondro-stimulative effect, and at least one filling material, in whichthe composition is in the form of a lyophil. In general, the at leastone differentiation or growth factor is a protein or peptide, preferablya glycoprotein or glycopeptide, preferably a cytokine, for example thedifferentiation or growth factor is a human differentiation or growthfactor. In a related embodiment, the differentiation or growth factor isrecombinantly produced. In general, the differentiation or growth factorcomprises at least one member of the TGF (Transforming Growth Factor)family. For example, the differentiation or growth factor comprises atleast one member of the group of BMP (Bone Morphogenetic Proteins),preferably BMP-2 and/or BMP-7.

Alternatively, the differentiation or growth factor is a TGF-β(Transforming Growth Factor beta) or is a VEGF (Vascular EndothelialGrowth Factor).

The composition in certain embodiments includes, the differentiation orgrowth factor in relation to the amount of collagen, osteo-inductive orchondro-inductive activity, as a share of about 0.005 to about 5 percentby weight, preferably from about 0.01 to about 4 percent by weight. Forexample, the differentiation or growth factor comprises a share of about0.05 to about 2 percent by weight, preferably from about 0.1 to about 5percent by weight, preferably firm about 0.5 to about 3 percent byweight. The composition further includes at least one scaffold material,preferably a scaffold material having osteo-conductive properties. Forexample, the scaffold is collagen, preferably type I collagen.Alternatively, or in addition the scaffold material is at least oneselected from the group of ceramics, biologically compatible metals andbiologically compatible polymers. Alternatively, or in addition thescaffold material is an extract of native bone, preferably ademineralized bone matrix (DBM). Alternatively the composition includesa growth factor having chondro-inductive properties, such as TFG-β.

In certain embodiments, the composition further includes at least oneadditional active substance, preferably at least one cytostatic and/orat least one antibiotic. For example the composition includes further atleast one component selected from the group of: at least one recruitingfactor, at least one adhesion factor, at least one growth factor and atleast one maturation factor for osteogenesis or chondrogenesis.

In general, the collagen is of mammalian origin, preferably of human,bovine, porcine or equine origin. In general, the composition furtherincludes pores, preferably interconnecting pores, preferably poreshaving a diameter in the range of about 100 μm to about 300 μm, and thecomposition has a sponge-like consistency. The pores can arise from alyophililization process, from a tunneling of ice crystal across athermal gradient, or from a granulation process. In general, thecomposition is at least partially bioresorbable, preferably completelybio-resorbable. The composition is produced in the form of a paste a gelor a lyophil. Accordingly, the composition has a substantiallyform-stable body.

In another embodiment, the invention provides a composition for treatingbone and/or cartilage defects having at least one recombinant collagen,preferably of human origin, and preferably containing at least onesubstance having an osteo-inductive or chondro-inductive activity, suchthat the osteo-inductive or chondro-inductive activity is at least onerecombinant bone morphogenesis protein (BMP). In general, the BMP is ofmammalian origin, preferably of equine, porcine or bovine origin. Forexample, the BMP is of human origin. Similarly, the BMP has an aminoacid sequence at least about 70% identical to a BMP of human origin. TheBMP in certain embodiments is recombinantly produced in a yeast or in abacterium. For example, the BMP is recombinantly produced in bacteriumEscherichia coli, or in Bacillus subtilis, or Streptomyces spp.Alternatively, the BMP is recombinantly produced in a cell capable ofprotein glycosylation. For example, the BMP is produced in a cellselected from the group consisting of mammalian, bird, yeast, andinsect.

In certain embodiments, the BMP, in relation to the amount of collagencomprises a proportion of about 0.005 to about 5 weight percent,preferably from 0.01 to 4 weight percent. The BMP comprises a proportionof about 0.05 to about 20 weight percent, preferably from about 0.1 toabout 5 weight percent, preferably from 0.5 to 3 weight percent.

In certain embodiments, the further additionally includes at least onescaffold material having osteo-conductive properties. For example, thescaffold material includes collagen, preferably type I collagen,preferably human type I collagen. Alternatively the collagen is typeIII. The scaffold material comprises at least one substance selectedfrom the group of tricalcium phosphate, ceramic materials, biologicallycompatible metals, and biologically compatible polymers. For example,the scaffold material is tricalcium phosphate or a biphasic materialcomprising tricalcium phosphate. These ceramics are made at differentsintering temperatures as is well known to one of ordinary skill in theceramics art. The composition can further include at least oneadditional active substance, preferably at least one cytostatic and/orat least one antibiotic. For example, the composition further includesat least one additional component having an activity selected from thegroup of at least one recruiting factor, at least one adhesion factor,at least one growth factor and at least one maturation factor forosteogenesis or chondrogenesis. In general the human collagen isrecombinantly produced.

The composition in general has a porous form, with pores, preferablyinterconnecting pores, preferably with diameters having a size in therange of about 100 μm to about 300 μm such that the composition has aspongy consistency. The pores arise from the lyophilization orgranulation process, as the composition is “non-woven” material.

The composition in general is substantially bioresorbable or isessentially completely bioresorbable. The composition is provided as alyophil, a gel, or a paste, and is a substantially form-stable body.

Thus, an embodiment of the invention provides a composition for treatingbone and/or cartilage defects comprising an aseptically preparedadmixture of a recombinant human collagen and a recombinant human bonemorphogenesis protein (BMP) such that the collagen and the BMP aresterile and are aseptically mixed and co-lyophilized or co-granulated.For example, the BMP has an amino acid sequence that is at least about70%, about 80%, about 85%, about 90%, or at least about 95% identical toan amino acid sequence of a BMP of human origin. The BMP in certainembodiments is produced in a bacterium, for example, the BMP is producedin E. coli. Alternatively, the BMP is recombinantly produced in a cellcapable of protein glycosylation. For example, the BMP is produced in acell selected from the group consisting of a mammalian, bird, yeast, andinsect cell. The BMP, in relation to the amount of collagen comprises aproportion of about 0.005 to about 5 weight percent, preferably from0.01 to 4 weight percent. For example, the BMP comprises a proportion ofabout 0.05 to about 2 weight percent, preferably from about 0.1 to about5 weight percent, preferably from about 0.5 to about 3 weight percent.

The invention in a further embodiment provides a method formanufacturing any of the above compositions, the method including:mixing components comprising at least one collagen, preferably of animalorigin and preferably having at least one osteo-inductive orchondro-inductive activity, with at least one differentiation or growthfactor having osteo-stimulative and/or chondro-stimulative activity, andco-lyophilizing the components to obtain an at least partly form-stable,spongy body. In an embodiment in which collagen is of non-human origin,the method further includes prior to mixing, contacting the collagenwith a peptidase or protease, preferably trypsin. The method furtherinvolves, prior to mixing, concentrating the differentiation or growthfactor, preferably in at least one separation step. A related embodimentof the method further includes prior to mixing, adding at least onescaffold material, such that the scaffold material has osteo-conductiveproperties and is selected from the group of ceramic materials,biologically compatible metals, biologically compatible polymers andextracts of native bone, preferably DBM, and mixing, preferably coating,with collagen; and mixing with at least one collagen, preferably ofanimal origin with an osteo-inductive or a chondro-inductive, activesubstance and/or at least one additive of at least one differentiationor growth factor with osteo-stimulative and/or chondro-stimulativeeffect; and colyophilizing the components to obtain an at least partlyform stable, spongy body. The method according to the above is performedunder aseptic conditions.

Also provided herein is use of at least one differentiation and/orgrowth factor with osteo-stimulative and/or chondro-stimulative effect,the use comprising mixing aseptically an aseptic or a sterile solutionof the growth factor with an aseptic or a sterile solution of at leastone collagen, preferably of animal origin and preferably having at leastone osteo-inductive or chondro-inductive, active substance; and,co-lyophilizing aseptically the resulting mixture in a container forsingle use as an implant in surgery.

Further provided herein is use of at least one human bone morphogenesisprotein (BMP) for treating a bone and/or cartilage defect during asurgical procedure, the use comprising adding the BMP to at least onehuman collagen and at least one scaffold material to obtain anadmixture, and providing the admixture as an aseptic device for treatinga bone and/or cartilage defect during a surgical procedure. The aboveuses are in the form of a device, which is provided as a surgery-readyunit dose. The composition is a lyophil, a gel or a paste.

Another embodiment herein provides a method for formulating acomposition for treating bone and cartilage defects, the methodcomprising: mixing aseptically a solution of a recombinant humancollagen with a solution of an amino acid sequence of a recombinanthuman bone morphogenesis protein; and granulating or lyophilizingaseptically the admixture to a gel, paste, or a lyophil having an atleast partly form-stable shape and a spongy consistency. In general, thesolution of the collagen and the solution of the bone morphogenesisprotein have reduced microbial content includes, or are sterile. Thusthe method includes, prior to the mixing, concentrating the collagen,preferably in at least one separation step, and the collagen is treatedwith acid or filter sterilized prior to or after concentrating. Furtherthe method can include prior to mixing, providing at least one scaffoldmaterial selected from the group of ceramic materials, biologicallycompatible metals, biologically compatible polymers and extracts ofnative bone, preferably DBM; mixing or preferably coating the scaffoldmaterial with the collagen; and colyophilizing or co-granulating theadmixture to obtain an at least partly form-stable, spongy body. Priorto mixing, the collagen is sterilized by filter sterilizing oracidifying, the scaffold material is irradiated, and mixing is performedunder aseptic conditions.

Yet another embodiment of the invention herein provides a kit fortreating a bone and/or cartilage defect during a surgical procedure, thekit having an aseptically prepared admixture in the form of a lyophil, agel or a paste, the admixture comprising a recombinant human bonemorphogenesis protein (BMP) and a recombinant human collagen, in acontainer. The kit is for example, such that the admixture is present ina surgery ready unit dose. In a related embodiment the kit further hasinstructions for direct use of the aseptic admixture as an implantwithout additional manipulation.

An embodiment of the invention herein provides an aseptic device forsurgical implantation for treating bone and/or cartilage defects whichhas an admixture of a recombinantly produced human differentiationand/or growth factor having osteo-stimulative and/or chondro-stimulativeeffect and at least one scaffold material, such that the factor is atleast about 70% identical in amino acid sequence to a human growthfactor, and the device is provided as a single component for surgicalimplantation. In one embodiment, the growth factor is glycosylated.Alternatively, the growth factor is recombinantly produced in a cell ofa yeast, a mammal or a bird. For example, the growth factor isnon-glycosylated, the growth factor is recombinantly produced in abacterium, for example, in E. coli.

In general the growth factor in the device is a member of the TGF(Transforming Growth Factor) family. For example, the growth factor isat least one of Bone Morphogenetic Proteins (BMP) comprising BMP-2,BMP-7, BMP-9, BMP-16; Vascular Endothelial Growth Factor (VEGF);Transforming Growth Factor β (TGFβ), Platelet Derived Growth Factor(PDGF), Insulin-like Growth Factor (IGF) and p15. In one particularembodiment, the growth factor is a human BMP-2 or a human BMP-7 that isrecombinantly produced and is nature identical. In general, theembodiments of the device that include collagen, the ratio of thedifferentiation or growth factor in relation to the amount of collagencomprises a share of about 0.005 to about 5 percent by weight,preferably from about 0.01 to about 4 percent by weight. For example,the growth factor comprises a share of about 0.05 to about 2 percent byweight, preferably from about 0.1 to about 5 percent by weight,preferably from about 0.5 to about 3 percent by weight.

The device in related embodiments includes at least one scaffoldmaterial, preferably a scaffold material having an osteo-conductiveactivity. For example, the scaffold is collagen having an amino acidsequence substantially identical to a human collagen. Further, thecollagen is recombinantly produced. Alternatively or in addiction, thescaffold material is at least one selected from the group of ceramics,biologically compatible metals and biologically compatible polymers. Thedevice can further include at least one additional active substanceselected from the group of at least one cytostatic agent and/or at leastone antibiotic. Alternatively, the device can further include at leastone component selected from the group of: a recruiting factor, anadhesion factor, a growth factor and a maturation factor forosteogenesis or chondrogenesis. The device in general is a co-lyophil ora co-granulate and is a spongy form-stable body that comprises pores.The pores have a diameter in the range of about 100 μm to about 300 μm,and such that the admixture has a consistency and stable form of asurgical sponge. The device admixture in related embodiments is at leastpartially bio-resorbable, and is preferably completely bio-resorbable.The admixture is generally aseptically prepared from aseptic or sterilecomponents. The device in various embodiments is provided in a closedcontainer. In a particular embodiment, the admixture is asepticallylyophilized in the container. For example, the container is asepticallycovered with a material capable of unidirectional passage of solventmolecules during lyophililization.

In general the scaffold material includes human recombinant collagen.For example, the collagen is selected from at least one of a type Icollagen and a type III collagen. Alternatively or in addition, thescaffold material comprises a mineral. For example, the mineral isselected from the group consisting of: tricalcium phosphate, ceramicmaterials, biologically compatible metals, and biologically compatiblepolymers. In certain embodiments, the device includes collagen that iscross-linked using a commonly known reagent for cross-linking proteins.For example, the collagen is covalently internally cross-linked toitself, or the collagen is covalently linked to the growth factor. Thusthe collagen is covalently cross-linked in solution to the growth factorto obtain a matrix, and then the matrix is lyophilized aseptically andused as an implant in a subject for treatment of a bone and/or acartilage defect.

Accordingly, following implant into the subject, the matrix issubsequently metabolized releasing slowly the covalently bound growthfactor such as BMP-2 or BMP-7, compared to release of an otherwiseidentically constituted and non-covalently bound matrix releasing growthfactor in a burst at an early time point following implant into thesubject. In certain embodiments of the device, the covalently crosslinked collagen is obtained by contacting the collagen and osteo- and/orchondro-stimulating factors with a bidentate, low molecularcross-linking reagent selected from the group of commonly used inprotein and peptide chemistry reagents:1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), succinicanhydride, a di-isocyanates, an activated and properly protected aminoacids, a double activated polyethylene-glycol, and an activated carbonhydrate to obtain quantitative binding of the reagent to the collagen.Accordingly, the device, following quantitative binding of the reagentto collagen is further prepared by the steps of: removing excess reagentby dialyzing, to obtain a modified collagen that retains proteinfeatures having a biological activity selected from the group ofosteo-inductive, chondro-inductive, osteo-conductive andchrondo-conductive; mixing the modified collagen with the factor/s for atime appropriate for cross-linking; and lyophilyzing, such that thesteps are performed aseptically. The device in embodiments aboveincludes materials that are aseptic, such that the device has a reducedmicrobial content.

In yet another embodiment, the device contains at least one additionalcomponent that retards release of the growth factor from the scaffold,for example from the collagen. The additional component retards releaseof the growth factor by non-covalent affinity, for example, theadditional component is a plasma protein such as fibrin or a globulin.The additional component is in related embodiments of human origin,and/or is recombinantly produced.

DETAILED DESCRIPTION

A problem in treatment of bone and cartilage defects is that certainsynthetic materials currently used to repair bone defects are poorlyabsorbed or are non-bioresorbable. These include ceramic materials, forexample porous calcium phosphate ceramics. Such materials have usefulosteo-conductive properties, i.e., the materials are able to provideregenerating bone tissue with a structure creating matrix (guidestructure) with sufficient mechanical stability. Bone generating cellsbind to materials with osteo-conductive properties, and then precipitatea bone matrix around or inside this material. Ceramic materials,however, remain in an unchanged state in the body, and are consequentlyovergrown and penetrated by the newly generated bone.

Bioresorbable bone replacement materials include synthetic polymers suchas polylactide and polyglycolide. Such synthetic materials are degradedin vivo, usually by hydrolysis. Degradation of these materials can leadto a release of acidic by-products such as lactic acid or glycolic acid,causing undesired local hyper-acidification in the body.

Bone replacement materials currently used to repair bone defects includeextracts of natural bone, such as bovine bone, porcine bone, or equinebone. Demineralized bone is a natural bone material in which theinorganic portion (mineral portion) has been removed and the remainingorganic matrix substantially retains the natural endogenous collagenwith other additional proteins. The extract retains a portion of thefunctional properties of the original bone. Further, these extracts ofbone are found to enhance repair of bone defects.

Bone replacement materials and methods of manufacturing thereof, areshown in for example WO 91/06324 A1 and WO 93/20857 A1, and arecommercially available products COLLOSS® and COLLOSS® F, which arecollagen extracts of bovine bone and equine (E) bone respectively. Theseproducts contain collagen type I of animal origin in combination withendogenous growth and differentiation factors retained and bound to thecollagen.

COLLOSS® and COLLOSS® E are provided in the form of a lyophilizedextract. These products show osteo-conductive and osteo-inductiveactivity, and have a further differentiating activity when contacted tostem cells. The term, “osteo-inductivity” as used herein means that thesubstance or material initiates regeneration of bone (osteogenesis) andstimulates regeneration of bone (osteo-stimulation), and thus promotesbone growth. The regeneration activity has been demonstrated in vivo,and also in cell culture ex vivo. Under certain circumstances, materialshaving these properties can be manipulated to amplify the regenerationprocess compared to normal bone growth. The osteo-conductive effects ofeach of the above products, however, are not sufficient for certainapplications.

Similar considerations apply to cartilage defects and to remedies forcartilage defects. The term, “chondro-inductivity” as used herein meansthat the substance or material initiates regeneration of cartilage(chondrogenesis) and stimulates regeneration of cartilage(chondro-stimulation), and thus promotes cartilage growth.

A complex for growth of bone tissue that includes a bone derivedcomponent, a bone derived chemotactic component, a bone derived adhesioncomponent, and a bone derived growth factor is shown in WO 91/06324 A1(corresponding to U.S. Pat. No. 5,932,207 issued Aug. 3, 1999) which isherein incorporated by reference in its entirety. This patent showsexamples of the adhesion component, fibronectin, tenascin, cytotactin,laminin, chondroinectin, collagen yes IV, V, VII, N-CAM, L-CAM, andintegrin, however without a filling agent that increases theosteo-conductive effect as well as an osteo-inductive effect of thecomplex.

Osteogenic compositions are shown in Chen et al. (U.S. Pat. Nos.5,707,962, issued Jan. 13, 1998, and 6,180,606, issued Jan. 30, 2001).Osteogenic devices are shown in Opperman et al. (U.S. Pat. Nos.5,958,441, issued Sep. 28, 1999, and 6,551,995, issued Apr. 22, 2003).

The compositions and devices provided herein contain a structuralcomponent having osteo-inductive and/or osteo-conductive properties,such as collagen, for example a collagen of animal origin. The animalorigin of the collagen contained in the composition and device providedherein is not limiting. In certain embodiments of the device andcomposition herein, the collagen of the composition is of human origin,and contains at least one osteo-inductive or chondro-inductive activesubstance of human origin. In alternative embodiments, the collagen isof nonhuman animal origin, for example of bovine, porcine or equineorigin.

The devices and compositions further include at least one additive of atleast one differentiation and/or growth factor, having osteo-stimulativeand/or chondro-stimulative activity. These differentiation and/or growthfactors are, for example, proteins or peptides, such as cytokines,glycoproteins or glycopeptides.

In certain embodiments, the differentiation and/or growth factors areprimarily human differentiation or growth factors and are combined withhuman collagen. In alternative embodiments, the human differentiation orgrowth factor is combined in the device with a collagen of xenogenicorigin (in relation to human), i.e., of non-human animal origin, forexample, bovine, porcine or equine origin.

In general, the differentiation or growth factor of human or animalorigin is recombinantly produced. The term “recombinant” refers toproteins produced by manipulation of genetically modified organisms, forexample micro-organisms.

In accordance with the present invention, polynucleotide sequences thatencode differentiation or growth factors are used in recombinant DNAmolecules to direct the expression of the differentiation or growthfactors in appropriate host cells. To express a biologically activehuman differentiation or growth factor, a nucleotide sequence encodingthe differentiation or growth factor, or functional equivalent, isinserted into an appropriate expression vector, i.e., a vector thatcontains the necessary nucleic acid encoding elements that regulatetranscription and translation of the inserted coding sequence.

Methods that are well known to those skilled in the art are used toconstruct expression vectors containing a sequence encoding thedifferentiation or growth factor and appropriate transcriptional ortranslational controls. These methods include in vitro recombinant DNAtechniques, synthetic techniques and in vivo recombination or geneticrecombination. Introduction of deletions, additions, or substitutions isachieved using any known technique in the art e.g., using PCR basedmutagenesis. Such techniques are described in Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.,1989 and Ausubel et al., Current Protocols in Molecular Biology, JohnWiley & Sons, New York, N.Y., 1989.

A variety of expression vector/host systems are utilized to contain andexpress a human differentiation or growth factor encoding sequence.These include but are not limited to microorganisms such as bacteriatransformed with recombinant bacteriophage, plasmid or cosmid DNAexpression vectors; yeast transformed with yeast expression vectors;insect cell systems infected with virus expression vectors (e.g.,baculovirus); plant cell systems transfected with virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with bacterial expression vectors (e.g., Ti, pBR322,or pET25b plasmid); or animal cell systems. Alternatively, chemicalmethods are used to synthesize the differentiation or growth factoramino acid sequence, whole or in part. For example, peptide synthesiscan be performed using various solid-phase techniques (Roberge et al.,Science 269:202, 1995) and automated synthesis may be achieved, forexample, using the 431A peptide synthesizer (available from AppliedBiosystems of Foster City, Calif.) in accordance with the instructionsprovided by the manufacturer.

The differentiation or growth factor in another embodiment is at leastone member of the TGF (Transforming Growth Factor) family. TGF familygrowth factors include, for example, various cytokines such as membersof the TGF-β family, that include the BMP family (Bone MorphogeneticProteins), for example, BMP-2 and BMP-7. In certain embodiments, thedifferentiation and/or growth factor is BMP granulated with for examplepolylactide (micro capsules).

BMP compositions of human origin and of bovine origin, and processes ofisolating BMP compositions and factors are shown in Urist (U.S. Pat. No.4,619,989, issued Oct. 28, 1986).

For BMP-2, the nucleic acid sequence, amino acid sequence, structure,and composition of BMP-2, and methods of manufacture are shown in Wanget al. (U.S. Pat. No. 5,631,142, issued May 20, 1997). Purified BMP-2and BMP-4 proteins and processes for producing them are shown in Wang etal. (U.S. patent application number 2007/0026437, published Feb. 1,2007). Methods of production of recombinant BMP-2 are shown in Rainer etal. (U.S. patent application number 20040018595, published Jan. 29,2004).

Recombinant human BMP-2 is also commercially available from ShenandoahBiotechnology, Inc. (Warwick, Pa.).

Purified BMP-7 proteins and processes for producing them are shown inWozney et al. (U.S. Pat. No. 5,366,875, issued Nov. 22, 1994). DNAsequences encoding BMP-7 proteins are shown in Rosen et al. (U.S. Pat.No. 5,141,905, issued Aug. 25, 1992). Recombinant human BMP-7 is alsocommercially available from Shenandoah Biotechnology, Inc. (Warwick,Pa.).

Each of the above references is incorporated by reference in itsentirety.

Additional suitable differentiation or growth factors that are used incompositions and devices herein include VEGF (Vascular EndothelialGrowth Factor), IGF1 (Insulin Growth Factor 1), FGF (Fibroblast GrowthFactor) and PDGF (Platelet Derived Growth Factor). Alternatively, afunctional fragment of any of the above mentioned differentiation orgrowth factor is used. The term “functional fragment” refers tomolecules of a modified structure and/or sequence that provide the samebiological function as the above mentioned differentiation or growthfactors. Functional fragments include proteins that contain naturallyoccurring or engineered alterations, including any of a deletion, anaddition, a substitution or other modification. A functional fragment isexemplified by a protein having a naturally occurring sequence for aregion of any of the above mentioned differentiation or growth factorsby a corresponding region of a growth factor from another mammalianspecies, including but not limited to, bovine, canine, feline, caprine,ovine, porcine, murine, and equine species.

The term “substantially identical” is used herein to refer to a firstamino acid sequence that contains a sufficient or minimum number ofamino acid residues that are identical to aligned amino acid residues ina second amino acid sequence such that the first and second amino acidsequences can have a common structural domain and/or common functionalactivity. For example, amino acid sequences that contain a commonstructural domain having at least about 60%, or 65% identity, preferablyat least 75% identity, more preferably at least 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identity.

Calculations of sequence identity between sequences are performed asfollows. To determine the percent identity of two amino acid sequences,the sequences are aligned for optimal comparison purposes (e.g., gapscan be introduced in one or both of a first and a second amino acidsequence for optimal alignment). The amino acid residues atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the proteins are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences are accomplished using a mathematical algorithm.Percent identity between two amino acid sequences is determined using analignment software program using the default parameters. Suitableprograms include, for example, CLUSTAL W by Thompson et al., Nuc. AcidsResearch 22:4673, 1994 (www.ebi.ac.uk/clustalw), BL2SEQ by Tatusova andMadden, FEMS Microbiol. Lett. 174:247, 1999(www.nobi.nlm.nih.gov/blast/bl2seq/bl2. html), SAGA by Notredame andHiggins, Nuc. Acids Research 24:1515, 1996(igs-server.cnrs-mrs.fr/˜cnotred), and DIALIGN by Morgenstern et al.,Bioinformatics 14:290, 1998 (bibiserv.techfak.uni-bielefeld.de/dialign).

In certain embodiments, the device or composition provided herein isengineered to provide for slow time-release of the active agents. Forexample, the device or composition further includes at least oneadditional component capable of retarding release of the differentiationor growth factor from the composition. Retarding release components are,for example a “sticky” protein capable of substantial nonspecificbinding, such as a protamine and a plasma protein such as globulin,albumin, and fibrin. Protamine is an alkaline protein extracted fromfish sperm and has a molecular weight of about 5000 Daltons. In analternative embodiment for retarding release, the growth ordifferentiation factor is covalently bound to the collagen, for exampleby a protein cross-linking agent.

In certain embodiments of the invention provided herein, the device orcomposition further includes at least one filler or filling material.The filling or filler material has several purposes, such as providingthe composition with additional mechanical stability and/or stability ofform or shape. In general, the filling material has osteo-conductiveproperties, and provides additional surfaces for cell proliferationand/or cell differentiation.

The devices or compositions that contain filling materials such ascollagen and at least one osteo-inductive or chondro-inductive activesubstance are manufactured to specific quantities of each component inthe composition. Relative to the amount of collagen, the osteo-inductiveor chondro-inductive active substance is contained in an amount of about0.005 to about 5.0 mass percent, for example about 0.01 to about 4.0mass percent. In other embodiments, the additive is contained in anamount of about 0.05 to about 2.0 mass percent, for example about 0.1 toabout 5.0 mass percent or about 0.5 to about 3.0 mass percent.

The filling material is typically collagen, such as type I collagen. Thecollagen is obtained from skin, tendon, and/or from bone tissue. Incertain embodiments, the collagen is of non-human origin, and the tendonis animal tendon, such as equine tendon. In alternative embodiments, thecollagen is a recombinantly produced collagen, for example,recombinantly produced human collagen. Further, non-collagen fillingmaterials are within the scope of the compositions and devices herein.Such filling materials are usually contained in the devices herein inthe same amounts and proportions as the collagen relative to theosteo-inductive or chondro-inductive active substance.

Additional filling materials suitable to the compositions and devicesherein include ceramic materials, biologically compatible metals, andbiologically compatible polymers. Ceramic materials includetricalciumphosphate (TCP), hydroxyapatite (HA), calcium sulfate, CMC(carboxylmethyl cellulose), sepharose, polyarylate, and mixtures of suchmaterials. In certain embodiments, the filling material is aβ-tricalciumphosphate. Such filling materials are, present in thecompositions and devices herein in relation to the total volume of theremaining elements, at least about 50 volume percent, at least about 80volume percent, at least about 95 volume percent, or at least about 99volume percent.

Ceramic filling materials are useful because the ionogenic elements ofsuch materials, i.e., the fixed group of atoms in an ion exchanger thatare either ionized or capable thereof, constitute the main elements ofnatural bone and mineral bone substance, and are therefore suitablebio-mimetic materials.

Ceramic material is used as a filling material in the commerciallyavailable product OSSAPLAST® (commercially available from Ossacur,Stuttgart, Germany). This product includes a bioresorbable andgranulated β-tricalciumphosphate with a high interconnecting porosityand a large specific surface.

Filling material is further exemplified by a biologically compatiblemetal. Examples of biologically compatible metals are magnesium,titanium and tantalum. Further, magnesium is also bioresorbable.Examples of biologically compatible polymers include synthetic polymerssuch as polylactide and polyglycolide. The filler agent in otherembodiments is a derivative of native bone, for example, demineralizedbone matrix (DBM).

In certain embodiments, the filler agent provides additionalosteo-inductive or chondro-inductive activity to the composition and/ordevice herein. These filler materials include leukotrienes, cytotactin,tenascin, laminin, fibronectin and cytokines such as BMP-1, BMP-2,BMP-7, IGF-1, TGF-β1, FGF and PDGF.

In embodiments of methods of manufacture of the devices andcompositions, the filler material is added to a collagen matrix bymodifying the collagen prior to the addition. For example, the collagenis chemically modified so that the filling material is covalentlycross-linked to the collagen. In an alternative embodiment, the fillingmaterial is added prior to preparation of the collagen matrix, or is anaturally occurring factor in the corresponding collagen matrix, i.e.,the collagen and the filling material are combined by the process bywhich they are provided, for example, the growth factor is extractedfrom its biological source, viz., natural bone. In this embodiment, theosteo-inductive or chondro-inductive filling materials are present in anative form in association with the collagen.

One or more additional agents that provide a further desired biologicaleffect with respect to treating bone or cartilage defects is added inother embodiments of the devices and compositions provided herein. Suchadditional agents are, for example, a cytostatic agent or an antibiotic,or a combination thereof.

In another embodiment, the device or composition further includesrecruiting factors, for example a chemotactic agent (such as achemotaxin or a leucotriene). The recruiting factor induces aninfiltration of the composition into cells of a human, such asmesenchymal stem cells, cartilage premonitory stem cells, fibroblastsand/or thrombocytes. Alternatively these substances are added to thecollagen, or are supplemented.

In another embodiment, the collagen contained in the composition furtherincludes at least one adhesion factor, for example cytotactin, tenascin,laminin and/or fibronectin. These substances function to fix recruitedor migrant cells in the composition or device according to the inventionat its application location (implant location).

In certain embodiments, the above mentioned factors are naturally foundin collagen, such that the filling material can be purified, or can beobtained in a complex that is a mixture of a plurality of these factors.The collagen component of the composition or device can include asubstantial portion of the osteo-inductive properties of the fillingmaterials that are involved in vivo for regeneration of bone and/orcartilage. A set of the naturally occurring filling materials mentionedabove can have overlapping or redundant active functions, such that apartial or substantial loss of activity of one or more active substancesmay be compensated by the presence of other active substances in thecomposition.

Collagen of animal origin is commercially available in isolation form,e.g., COLLOSS® (bovine) and COLLOSS® E (equine) from Ossacur(Oberstenfeld, Germany). These products contain native activesubstances, so that the combination of the collagen and active substanceprovides a native, functional unit, the composition corresponding in aparticular manner to the in vivo milieu, in which bone and/or cartilageregeneration is induced.

Methods of extracting collagen from natural bone are well known in theart. Collagen is extracted from natural bone by the following exemplaryprocedure: pulverizing natural bone; degreasing by extraction with anorganic solvent; demineralizing with acid treatment, for examplehydrochloric acid; incubating with chelating agents, for exampleethylenediamine tetraacetic acid (EDTA) or2-amino-2-hydroxymethyl-1,3-propanediol (TRIS) or a combination thereof;and extracting with guanidine, for example guanidine hydrochloride;followed by purifying the extract; and physically forming the extractinto a configuration suitable for implanting. One of ordinary skill inthe art of biology will understand that one or more of the above stepsmay be omitted as necessary or additional steps are within exemplaryprocedures.

In certain embodiments, the composition or device is in a porousconfiguration or form. In other embodiments, the device or compositionhas a substantially spongy consistency. The type filling material, asdescribed above such as, ceramic materials, e.g., tricalciumphosphate(TCP), determines the extent of porosity of the composition or device.

In certain embodiments, the porosity is an interconnecting porosity,i.e., the pore structure is located between the individual particles ofthe filling material, and within the particles. A porous device orcomposition is advantageous for cell infiltration from cells of thesubject recipient of the implant into the pores of the device orcomposition, for example bone and/or cartilage cells, or progenitorcells thereof.

Pores of the composition or device or the pores of the filling material,respectively, have diameters of at least about 100 μm, particularlydiameters of about 100 μm to about 300 μm. In certain embodiments, thepores of the composition or device or the pores of the filling material,respectively are about 200 μm in diameter.

In certain embodiments, the composition and device according to theinvention is at least partly bio-resorbable. In a related embodiment,the composition and device according to the invention is completelybioresorbable. Following implantation, the implant to treat the bone orcartilage defect is completely replaced by new bone or new cartilage.

In another embodiment, the composition according to the invention is alyophil, for example colyophil. The advantages of a lyophil aredescribed in the Examples below.

In another embodiment, the composition according to the invention is asubstantially form-stable body, for example, a porous spongy structurewith sufficient mechanical stability to withstand mechanicalmanipulation involved with implantation. Such a composition includesosteo-inductive/chondro-inductive properties for regeneration of bone orcartilage.

In general, the composition and/or device provided herein is produced inan aseptic or sterile procedure and is packed in a sterile procedure.Further, the device is unitary and requires no manipulation so it can beimplanted directly for the treatment of the bone or cartilage defectwithout additional preparation.

Another aspect of the present invention provides a new method formanufacturing the above described composition according to theinvention. The method involves providing a collagen, for example acollagen of human origin, recombinant collagen, or a collagen of animalorigin, which includes at least one osteo-inductive or chondro-inductiveactive substance; mixing this collagen with an additive of at least onedifferentiation or growth factor with osteo-stimulative and/orchondro-stimulative effect and possibly other substances; andcolyophilizing these components to form an at least partially stablespongy body.

In certain embodiments of the method according to the invention, priorto mixing the collagen with the at least one additive, the collagen istreated with at least one peptidase, for example an endopeptidase suchas trypsin. Treating with an endopeptidase removes undesired proteins orpeptides, for example telopeptides that are found in the collagen (withor without active substance), are removed from this collagen fraction.In general, the endopeptidase used in the described treatment, such astrypsin, is provided in such an amount that it is depleted or completelyremoved before affecting the osteo-conductive activity of the collagenfraction. One of ordinary skill in the art of biochemistry determinesappropriate concentrations of the endopeptidase and duration oftreatment to the mixture that does not affect the activity of thecollagen fraction. In other embodiments, collagen, for example type Icollagen, is not digested by an endopeptidase.

In other embodiments of the method, the collagen is concentrated priorto mixing the collagen with the additional one or more compounds.Concentrating the collagen prior to mixing the collagen with additionalcomponents is beneficial under certain circumstances with respect toproperties of the resulting colyophilized spongy body. Concentrating thecollagen fraction with added osteo-inductive or chondro-inductive activesubstances provides that the concentration of the active substances inthe total device having the collagen fraction together with the activesubstances of the additive, is great enough to substantially provide asufficient concentration of the active substances within the totalvolume of the composition, for example, the spongy body.

Concentrating a mixture having endogenous or added osteo-inductive orchondro-inductive active substances is accomplished by methods wellknown to one of ordinary skill in the art in biology. For example,concentrating involves at least one centrifuging step, at least oneseparation step through a suitable membrane, and/or at least oneprecipitation step by means of gravity. An exemplary concentration of aprotein-containing suspension of the product COLLOSS® E with a drysubstance content (TS) of 1.5 mg protein/g to 2.0 mg protein/gsuspension. The above described methods result in an increased TScontent between 2.0 mg protein/g to 20.0 mg protein/g suspension.

In general, a resulting spongy body provides the user with a homogeneousdistribution of the biologically active substances that form the spongybody. In general, the filling materials constitute a larger volume ofthe spongy body than the biological materials. The biological materialsobtained from collagen and from added agents such as differentiation orgrowth factors only cover the volume between the particles of thefilling materials.

In certain embodiments, collagen is mixed with a porous filling materialwith osteo-conductive properties, as described above. An exemplaryprocess for obtaining collagen with osteo-conductive properties involvesmixing collagen and an above mentioned filling material withosteo-conductive properties. During this mixing, the collagen is appliedas a coating on the inner and outer surfaces of the porous fillingmaterial with osteo-conductive properties. Further steps includelyophilizing the resulting mixture to obtain collagen withosteo-conductive properties, i.e., collagen having anosteo-inductive/osteo-stimulative orchondro-inductive/chondro-stimulative effect. The method furtherprovides at least one collagen with osteo-inductive orchondro-inductive, active substance and/or an additive of at least onedifferentiation or growth factor with osteo-stimulative and/orchondro-stimulative effect. The resulting mix is finally colyophilizedto form a substantially stable spongy body.

In general, the method described above is performed under asepticconditions, yielding a resulting device or composition according to theinvention that is in aseptic or even sterile. In alternativeembodiments, a variety of sterilization methods are used to obtain thecomposition that is aseptic or is sterile. In one embodiment, prior touse in the method according to the invention, each individual componentof the composition according to the invention is sterilized, forexample, filtered under sterile conditions to remove bacteria andviruses. In another embodiment, the method further includes packagingthe composition or device in a sterile manner to provide an asepticcomposition or device or a sterile composition or device.

The characteristics of the composition according to the invention shownherein are explicitly made characteristics of the method according tothe invention and the use according to the invention, respectively.

In the devices provided herein, presence of at least one differentiationand/or growth factor with osteo-stimulative and/or chondro-stimulativeeffect with at least one collagen results in several advantages, asshown below. Each of the agents induces and also amplifies theosteogenic or chondrogenic properties of the device of the presentinvention. Further, presence of growth factor with the collagen resultsin amplification of already existing form stability of the composition.The composition herein is optimized by interaction between theosteo-inductive and osteo-conductive effect provided by the growthfactor, and the form stability/supporting function provided by thefilling material. In another embodiment, any reduction ofosteo-inductive effect of the composition resulting from addition offilling materials that are not osteo-inductive is compensated orovercome by relatively small increases in the amount of the additive tothe composition.

Further, colyophilization in the production process, produces afinalized composition, i.e., the components of the composition arealready mixed to form a ready-to-use device. Another embodiment providesa ready-to-use device that is convenient for use, for example by asurgeon. Because the composition is produced in a sterile manner, it canbe taken directly from a sterile package and used during surgery torepair a bone or cartilage defect.

The invention having now been fully described, it is further illustratedby the following examples and claims, which are illustrative and are notmeant to be further limiting. Those skilled in the art will recognize orbe able to ascertain using no more than routine experimentation,numerous equivalents to the specific procedures described herein. Suchequivalents are within the scope of the present invention and claims.The contents of all references, including issued patents and publishedpatent applications cited throughout this application, are herebyincorporated by reference.

EXAMPLES Example 1 Production of Compositions According to the Invention

The following method is used for production of the compositionsaccording to the invention.

The product COLLOSS® E, a collagen extract of equine bone tissue, iscommercially available as described above. Methods of making thisproduct are also described above and are shown in WO 93/20857 A1, whichis herein incorporated by reference in its entirety. See also Spine30:890-895, 2005. This material contains an active substance complex ofnaturally bound factors in a matrix of type I collagen.

COLLOSS® E is available in the form of an aqueous suspension, which isconcentrated by performing at least one separation step. The suspensionobtained from the concentration is then mixed with further additives andelements of the composition. After obtaining a homogenous mixture, theelements of this mixture are colyophilised. A porous, spongy, stablebody occurs.

To obtain a sterile or aseptic device, the entirety of the steps in theproduction process are performed under aseptic conditions, and theindividual components of the composition are filtered in a sterilemanner in advance of their use in the manufacturing process.

Example 2 Compositions of the Invention

Compositions according to the invention are prepared from the followingelements:

No. Collagen matrix Additive Further additives 1 5 mg COLLOSS E 0.01 mghuman — BMP-2 (or BMP-7) 2 5 mg COLLOSS E 2 mg human BMP-2 — (or BMP-7)3 100 mg COLLOSS E 0.01 mg human — BMP-2 (or BMP-7) 4 100 mg COLLOSS E 2mg human BMP-2 — (or BMP-7) 5 No. 1-No. 4 No. 1-No. 4 1 ml TCP*(OSSAPLAST) 6 No. 1-No. 4 No. 1-No. 4 1 ml TCP (OSSAPLAST) 5 mg collagentype I 7 No. 1-No. 4 No. 1-No. 4 1 ml TCP (OSSAPLAST) 100 mg collagentype I 8 — 0.01 mg human 1 ml TCP (OSSAPLAST) BMP-2 (or BMP-7) 5-100 mgcollagen type I 9 — 2 mg human 1 ml TCP (OSSAPLAST) BMP-2 (or BMP-7)5-100 mg collagen type I *Tricalcium phosphate

Further examples of agents that, in certain embodiments are present inthe devices and compositions of the invention include: BMP colyophilizedwith collagen and/or ceramic; BMP bound with a spacer (globulin,polyethylene glycol (PEG), albumin, protamine, and fibrin) to thecollagen; and a suspension of collagen with a protamine, a globulin, afibrin, an albumin, and BMP and the suspension is then lyophilized.

The BMP-2 or BMP-7 or other growth factors in the device or compositionare for example a recombinantly produced human protein. The collagentype I used is for example a recombinant human collagen. Use ofrecombinant human BMP-2 or BMP-7 and recombinant human collagen isadvantageous when administering these compositions to human beingsbecause of severe antigenic activity of xenobiotic proteins.

1. A composition for treating bone and/or cartilage defects, comprisingat least one recombinant collagen wherein the recombinant collagen ischaracterized in that it is at least of animal origin, such asmammalian, preferably human, bovine, porcine, or equine; and at leastone substance having an osteo-inductive or chondro-inductive activity,at least one differentiation and/or growth factor havingosteo-stimulative and/or chondro-stimulative effect, wherein thedifferentiation or growth factor is characterized in that it is at leastone of: a protein or peptide; a glycoprotein or glycopeptides; acytokine; a human differentiation or growth factor; is recombinantlyproduced; and is at least one selected from the group of a ofTransforming Growth Factor (TGF) family including TGF P and a VascularEndothelial Growth Factor (VEGF); and a Bone Morphogenetic Protein (BMP)including BMP-2 and/or BMP-7; and at least one filling material, whereinthe composition is in the form of a lyophil and has a sponge-likeconsistency, is at least partially bioresorbable, and comprises asubstantially form-stable body.
 2. The composition according to claim 1,wherein the differentiation or growth factor in relation to the amountof collagen, osteo-inductive or chondro-inductive activity, comprises apercent share by weight in a range selected from: from about 0.005 toabout 5 about 0.01 to about 4, about 0.05 to about 2, about 0.1 to about5, and from about 0.5 to about 3 percent by weight.
 3. The compositionaccording to claim 1 further comprising at least one scaffold materialfurther characterized in that the scaffold material comprises at leastone of: a material having osteo-conductive properties, a collagen; atype I collagen; a recombinant collagen; at least one of a ceramic, abiologically compatible metal and a biologically compatible polymer; aplurality of pores; and ores having a diameter of about 100 μm to about300 μm.
 4. The composition according to claim 1, wherein the compositionfurther comprises at least one additional active substance selected fromthe group of, a cytostatic; an antibiotic; a recruiting factor; anadhesion factor; a growth factor; and a maturation factor forosteogenesis or chondrogenesis.
 5. A composition for treating boneand/or cartilage defects comprising at least one recombinant collagen ofhuman origin, and at least one recombinant bone morphogenesis protein(BMP) having an osteo-inductive or chondro-inductive activity, whereinthe BMP, in relation to the amount of collagen comprises a proportion ina range selected from the group of about 0.005 to about 5 weightpercent, about 0.01 to about 4 weight percent, about 0.05 to about 2weight percent, from about 0.1 to about 5 weight percent, and from about0.5 to about 3 weight percent wherein the recombinant BMP is ofmammalian origin, preferably of human, equine, porcine or bovine origin.6. The composition according to claim 5, further additionally comprisingat least one scaffold material having osteo-conductive propertieswherein the scaffold material comprises at least one selected from thegroup of: recombinant collagen, type I collagen, human type I collagen,tricalcium phosphate, a biphasic material comprising tricalciumphosphate, a ceramic material, a biologically compatible metal, and abiologically compatible polymer, wherein the composition comprises aporous form with pores, preferably interconnecting pores, havingdiameters in the range of about 100 μm to about 300 μm wherein thecomposition has a spongy consistency.
 7. The composition according toclaim 5, further comprising at least one additional active substanceselected from a cytostatic and an antibiotic, a recruiting factor, anadhesion factor, a growth factor and a maturation factor forosteogenesis or chondrogenesis.
 8. A composition for treating boneand/or cartilage defects comprising an aseptically prepared admixture ofa recombinant human collagen and a recombinant human bone morphogenesisprotein (BMP) wherein the collagen and the BMP are sterile and areaseptically mixed and co-lyophilized or co-granulated, wherein the BMPhas an amino acid sequence that is at least about 70%, about 80%, about85%, about 90%, or at least about 95% identical to an amino acidsequence of a BMP of human origin, wherein the BMP is produced in abacterium and is non-glycosylated or in a cell selected from the groupof mammalian, bird, yeast and insect and is glycosylated; wherein BMP inrelation to collagen comprises a proportion of weight percent selectedfrom the group of: about 0.005 to about 5; about 0.01 to about 4; about0.05 to about 2; about 0.1 to about 5; and about 0.5 to about 3 weightpercent
 9. A method for aseptically manufacturing the compositionaccording to claim 1, the method comprising: contacting the collagenwith a peptidase or protease, preferably trypsin, wherein the collagenis of nonhuman origin and has at least one osteo-inductive orchondro-inductive activity, adding at least one scaffold material,wherein the scaffold material has osteo-conductive properties and isselected from the group of ceramic materials, biologically compatiblemetals, biologically compatible polymers and extracts of native bone,preferably DBM, and mixing, preferably coating, with collagen, mixingcomponents comprising the collagen, wherein the collagen is of animalorigin and has at least one osteo-inductive or chondro-inductiveactivity, with the at least one differentiation or growth factor havingosteo-stimulative and/or chondro-stimulative activity and the scaffold,and co-lyophilizing the components to obtain an at least partlyform-stable, spongy body, wherein the method is performed under asepticconditions.
 10. A use of at least one differentiation and/or growthfactor with osteo-stimulative and/or chondro-stimulative effect the usecomprising mixing aseptically a sterile solution of the growth factorwith a sterile solution of at least one collagen, preferably of animalorigin and preferably having at least one osteo-inductive orchondro-inductive, active substance; and, co-lyophilizing asepticallythe resulting mixture in a container for single use as an implant insurgery.
 11. A use of at least one human bone morphogenesis protein(BMP) for treating a bone and/or cartilage defect during a surgicalprocedure, the use comprising adding the BMP to at least one humancollagen and at least one scaffold material to obtain an admixture,wherein the admixture is a lyophil, a gel or a paste, and providing theadmixture as an aseptic device for treating a bone and/or cartilagedefect during a surgical procedure, wherein the device is provided as asurgery-ready unit dose.
 12. A method for formulating a composition fortreating bone and cartilage defects, the method comprising:concentrating a solution of recombinant human collagen, wherein thecollagen is treated with acid or is filter sterilized; mixingaseptically the recombinant human collagen with a solution of arecombinant human bone morphogenesis protein, wherein the collagen andbone morphogenesis protein are sterile or have a reduced microbialcontent; and granulating or lyophilizing aseptically the admixture to agel, paste, or a lyophil having an at least partly form-stable shape anda spongy consistency.
 13. The method according to claim 12, furthercomprising prior to mixing, providing at least one scaffold materialselected from the group of ceramic materials, biologically compatiblemetals, biologically compatible polymers and extracts of native bone,preferably DBM; mixing or preferably coating the scaffold material withthe collagen, wherein the scaffold material is irradiated prior tomixing with the collagen; and colyophilizing, or co-granulating theadmixture to obtain an at least partly form-stable, spongy body.
 14. Akit for treating a bone and/or cartilage defect during a surgicalprocedure comprising an aseptically prepared admixture in the form of alyophil, a gel or a paste, the admixture comprising a recombinant humanbone morphogenesis protein (BMP) and a recombinant human collagen, in acontainers wherein the admixture is present in a surgery-ready unitdose, and further comprising instructions for direct use of the asepticadmixture as an implant without additional manipulation.
 15. An asepticdevice for surgical implantation for treating bone and/or cartilagedefects comprising an admixture of a recombinantly produced humandifferentiation and/or growth factor having osteo-stimulative and/orchondro-stimulative effect and at least one scaffold material, whereinthe factor is at least about 70% identical in amino acid sequence to ahuman growth factor, wherein the growth factor is at least one selectedfrom the group of Bone Morphogenetic Proteins (BMP) comprising BMP-2,BMP-7, BMP-9, BMP-16; Vascular Endothelial Growth Factor (VEGF);Transforming Growth Factor β (TGFβ), Platelet Derived Growth Factor(PDGF), Insulin-like Growth Factor (IGF) and p15, wherein the admixtureis aspetically prepared from aseptic or sterile components, wherein thedevice is provided as a single component for surgical implantation,wherein the device further comprises a closed container and theadmixture is lyophilized in the container.
 16. The device according toclaim 15, further comprising at least one scaffold material, wherein thematerial has an osteo-conductive activity wherein the scaffold isselected from the group consisting of: a collagen having an amino acidsequence substantially identical to a human collagen and isrecombinantly produced, a ceramic, a biologically compatible metal and abiologically compatible polymer.
 17. The device according to claim 15,further comprising at least one additional active substance selectedfrom the group of at least one cytostatic agent, an antibiotic, arecruiting factor, an adhesion factor, a growth factor, and a maturationfactor for osteogenesis or chondrogenesis.
 18. The device according toclaim 15, wherein the admixture is at least partially bio-resorbable,preferably completely bio-resorbable.
 19. The device according to claim15, wherein the container is aseptically covered with a material capableof unidirectional passage of solvent molecules during lyophilization.20. The device according to claim 15 comprising materials that areaseptic, wherein the device has a reduced microbial content.
 21. Thedevice according to claim 15, further comprising at least one additionalcomponent capable of retarding release of the growth factor from thescaffold, wherein the component is preferably a plasma protein such asfibrin.
 22. The device according to claim 16, wherein the scaffold iscollagen, and the collagen is cross-linked.
 23. The device according toclaim 22, wherein the collagen is covalently linked to the growth factoraseptically with a reagent commonly known to be used to cross-linkproteins selected from the group consisting of: bidentate low molecularcross-linking reagent selected from the group of commonly used inprotein and peptide chemistry reagents:1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), succinicanhydride, a di-isocyanates, an activated and properly protected aminoacids, a double activated polyethylene-glycol, and an activated carbonhydrate.
 24. The device according to claim 23, wherein followingimplanting into a subject, the device is subsequently metabolizedreleasing the covalently bound growth factor slowly, compared to releasein a burst at an early time point following implant into the subject ofan otherwise identically constituted and non-covalently bound growthfactor.
 25. The device according to claim 23, wherein followingquantitative binding of the reagent to collagen, the device is furtherprepared by the steps of: removing excess reagent by dialyzing to obtaina modified collagen that retains protein features having a biologicalactivity selected from the group of osteo-inductive, chondro-inductive,osteo-conductive and chrondo-conductive; mixing the modified collagenwith the growth factor/s for a time appropriate for cross-linking; andlyophilyzing, wherein the steps are performed aseptically.